1. Introduction
Fibroblast growth factor 2 (FGF2) is a member of the heparin-binding
growth factor (HBGF) family, and is widely expressed in development and
adult tissues (Nguyen et al. , 2013). FGF2 is a potent stimulator
of cell proliferation, differentiation, and migration of multiple cell
types, playing an essential role in embryonic development (Slacket al. , 1987), tissue repair (Maddaluno et al. , 2017), and
angiogenesis (Chu et al. , 2011; Corseaux et al. , 2000).
FGF2 can be used to accelerate the healing of both acute and chronic
wounds. However, it has a short circulation half-life due to rapidly
protease degradation, kidney filtration, and antigenic response,
limiting its clinical application. Thus, increasing the stability of
FGF2 is required to improve its application potential.
Although there are many chemical modification approaches to address
stability, poly (ethylene) glycol (PEG) modification has been well
demonstrated as an effective strategy to improve stability and
biocompatibility of proteins (Brocchini et al. , 2008; DeFreeset al. , 2006; Krall et al. , 2016). PEG is a substance that
has been designated by the Food and Drug Administration (FDA) as
“Generally Recognized as Safe (GRAS)” (Parnaud et al. , 1999),
and a variety of PEGylated proteins have been approved for use by FDA
(Dozier et al. , 2015). The most widely used PEG modification
method is to engineer a single cysteine into a protein, and then rapidly
and quantitatively react this cysteine with a PEG-maleimide group, thus
forming a protein-PEG conjugate (Foley et al. , 2007; Rosenet al. , 2017). Cysteine is an ideal target for site-specific
protein modification due to its typical low abundance in proteins and
the high nucleophilicity of the sulfhydryl side chain (Bernardimet al. , 2016). Previous efforts using site-selective PEGylation
of cysteine residues have resulted in modified proteins with improved
pharmacokinetics and retained biological activity (Dozier et al. ,
2015).
The crystal structure of the FGF2-FGFR-Heparin ternary complex shows
that FGF2 activity depends on the heparin-dependent formation of 2:2
FGF2-FGFR dimer complex (Beenken et al. , 2012). Heparin
facilitates FGF-FGFR dimerization by binding both FGF and FGFR, and
thereby promoting and stabilizing the protein-protein contacts between
ligand and receptor (Beenken et al. , 2009). Obviously, the
receptor binding region and heparin-binding region are important regions
for the activity of FGF2. However, there has been little detailed study
of structure-activity relationships in FGF2. Previous efforts to modify
FGF2 focused on the protein’s N-terminus or chemical modification of two
surface-exposed cysteines (Decker et al. , 2016; Kang et
al. , 2010), but the resulting long-acting FGF2 conjugates exhibited
reduced bioactivity. Therefore, a rational modification strategy based
on the structure to select optimal sites on the protein for cysteine
mutation may be more effective to obtain PEGylated proteins that retain
biological activity.
To identify suitable modification sites, four surface-exposed sites,
including two sites near the heparin- and FGFR-binding regions and two
native cysteines were selected and substituted by cysteine or alanine,
and PEG-FGF2 conjugates were synthesized and purified.
Structure-activity analysis and long-acting characteristics of these
conjugates were explored using in vitro and in vivo wound
healing models.